We have previously reported that the enzymic activity of rat and human liver 3-hydroxy-3-methylglutaryl Coenzyme A reductase (HMGR) is modulated in vitro in a bicyclic cascade system involving reversible phosphorylation of HMGR and reductase kinase. A calcium-activated and phospholipid-dependent protein kinase (protein kinase C) purified from rat brain cytosol is able to phosphorylate both insoluble microsomal (MW about 100,000) and purified soluble (MW 53,000) 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). The inhibition of enzymic activity and phosphorylation of HMGR was maximal at pH 6.5.. Lysine rich histone (type III-S) was the preferred substrate for protein kinase C. The phosphorylation of mixed casein was significantly increased by protein kinase C. The phosphorylation of phosvitin and protamine was not significantly stimulated by phospholipid and diolein. The phosphorylation and concomittant inactivation of enzymic activity of HMGR was absolutely dependent on calcium, phosphatidyl serine and diolein. Dephosphorylation of phosphorylated HMGR was associated with the loss of protein bound radioactivity and reactivation of enzymic activity. Maximal phosphorylation of purified HMGR was associated with the incorporation of 1.05 +- .016 mol of phosphate per mol of native form of HMGR (MW about 100,000). The apparent Km for purified HMGR and histone was .08 mg/ml, and .12 mg/ml, respectively. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA) stimulated the protein kinase C catalyzed phosphorylation of HMGR. Increased phosphorylation of HMGR by PMA suggests a possible in vivo protein kinase C mediated mechanism for the short-term regulation of HMGR activity. The identification of the protein kinase C system in addition to the reductase kinase-reductase kinase kinase bicyclic cascade system for the modulation of the enzymic activity of HMGR may provide new insights into the molecular mechanisms involved in the regulation of cholesterol biosynthesis.